Abstract
All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.
Highlights
All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression
T-box riboswitches use opposite strategies to control Met biosynthesis: SAM-I uses a negative feedback mechanism to turn off Met biosynthesis in response to increasing SAM concentration[14,15,17,18], while T-box uses a positive feedback mechanism to turn on Met biosynthesis in response to the accumulation of uncharged Met-tRNA8,11,12,19–22
Recent work examining the regulation of Met biosynthesis in the phytopathogen Xanthomonas campestris pv. campestris provided functional evidence of a Gram-negative bacterium utilizing a 5′ untranslated region (5′UTR) region to control the expression of the genes involved in the generation of Met[26]
Summary
All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Biochemical and genetic data demonstrate that SAM-IXcc expression platform can repress gene expression in response to SAM binding to SAM-IXcc aptamer and can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-ShineDalgarno (SD) sequence and freeing the SD for translation initiation These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNAsensing RNA element. Methionine (Met) is a unique proteinogenic amino acid which plays acritical role in the initiation of translation and the precursor of the principal cellular methyl group donor S-adenosylmethionine (SAM)[16] It has been shown in Gram-positive bacteria that the key regulators of Met biosynthesis are the SAM-I14,15,17,18 and T-box[8,11,12,19,20,21,22] riboswitches.
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